In previous studies we have established that plasma membranes of most cells contain adenosine receptors through which the nucleoside activates adenylate cyclase, but that the fat cell receptor mediates inhibition. With the use of dATP as the cyclase substrate, we now find that analogs, particularly N6-phenyliso-propyladenosine (PIA) and 5'-N-ethylcarboxamideadenosine (ECA), clearly distinguish between activatory and inhibitory receptors. Activation of liver and Leydig tumor cell cyclases exhibits the potency series: ECA greater that Adenosine greater than or equal to PIA, whereas the potency series for inhibition of the fat cell enzyme is: PIA greater than adenosine greater than PIA. Moreover, the respective potency series are maintained in physiologic studies with intact cells. These studies show that distinct subclasses of adenosine receptors exist, and underline the predictive value of the use of adenylate cyclase as a probe for development of pharmacologic agonists and antagonist of the adenosine receptor.